Glioblastoma multiforme (GBM) is the most common primary intracranial neoplasm and its almost uniform lethality. Temozolomide (TMZ) is the standard of care for the treatment of GBM patients showing small but significant increases in median survival of 4 weeks. However, acquisition of TMZ-chemoresistance is one of the major obstacles to efficient therapy for GBM patients. Despite aggressive treatment approaches, recurrence occurs in 90% of GBM patients. One cause of this poor outcome is development of a drug resistance. The mechanism underlying drug resistance is still not well understood thus challenging the development of more effective strategies and/or novel therapeutics to overcome this resistance. Mitochondria (mt) are at the cross road of energy production and apoptotic pathways. Their role in cancer etiology is significant; however, information regarding mt function and chemoresistance remains poorly defined. The enzyme cytochrome c oxidase (CcO) (EC 1.9.3.1) is a large mt transmembrane protein complex. It is the last enzyme in the respiratory electron transport chain (ETC) that transfers electrons from cytochrome c (Cyt c) to molecular oxygen (O2). Cyt c and O2 are the main substrates of CcO and both are directly involved in the intrinsic mt apoptotic pathway and hypoxia. While most of the studies to date have focus on CcO involvement in mt oxidative phosphorylation, any casual relation between CcO activity, CcO-nuclear-encoded subunits and the development of resistance to apoptosis in GBM is unclear and is the subject of this proposal. Recently, we demonstrated that acquisition of TMZ-resistance correlates with a significant increase of CcO activity in culture glioma cells as well a paired primary-recurrent GBM patient biopsies. The discovery of similar alterations in CcO activity between primary and recurrent human GBM specimens in patients subjected to TMZ-radiotherapy emphasizes the clinical significance of the findings and the primary role of TMZ in mediating these effects. Moreover, pharmacological or genetic inhibition of CcO in TMZ-resistant cells restores TMZ-induced apoptosis. The goal of this proposal is to test the hypothesis that CcO subunit 4 isoform 1 (COX4-1) drives chemoresistance in GBM through changes in 1) CcO assembly/ function and 2) cellular redox homeostasis by achieving three specific aims: We will determine whether: (1) Determine the effects of COX4-1 and 2 isoforms on the assembly/ function of CcO and on resistance to TMZ, (2) Determine the effects of COX4-1 and 2 isoforms on cellular redox status and on resistance to TMZ and 3) Determine the activity of CcO and expression of COX4-1 and 2 isoforms in brain tumor patient biopsies. Upon completion of this research, we expect to have developed a substantial base of knowledge on the role of CcO in the apoptotic response to TMZ. Our long term goal is to apply this information for the development of CcO-based therapies as well as for the development of prognostic markers for chemoresistance and GBM recurrence.